Genome-wide identification and molecular evolution of Dof transcription factors in Cyperus esculentus

Dof transcription factor family in Cyperus esculentus genome was identified and analyzed using bioinformatics. The analysis results revealed that C.esculentus genome contains 29 Dof genes (CesDof), all of which are located in the nucleus according to subcellular localization prediction. CesDof proteinrs have a range of 124 to 512 amino acids, with most being basic proteins. Their secondary structure was mainly irregular curl. The promoter sequence of CesDof genes contains cis-acting elements that respond to light, drought, hormones, low temperature, and circadian rhythm. Codon preference analysis showed that CesDof genes' codon preference ends in T/A. Collinearity analysis revealed that C.esculentus had three pairs of collinear CesDof genes. Additionally, there were 15 pairs of collinear genes between C.esculentus and Arabidopsis thaliana. The genetic relationship between C.esculentus and Rhynchospora pubera was found to be the closest. Phylogenetic tree analysis revealed that 29 CesDof genes of C.esculentus can be classified into 4 subgroups. Additionally, 144 miRNAs were predicted to target these CesDof genes. Furthermore, protein interaction analysis indicated that 15 Dof proteins in C.esculentus had interactions. The qRT-PCR verification results of drought stress and salt stress treatment experiments showed that most CesDof genes were involved in drought stress and salt stress responses, and the gene expression trends under drought stress and salt stress conditions were consistent. These results lay a theoretical foundation for further studying the molecular functions of Dof gene family in C.esculentus and its molecular mechanisms in regulating the life activities of C.esculentus. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10565-y.


Introduction
Cyperus esculentus is a plant belonging to the genus Cyperus in the family Cyperaceae.It is a masculine plant that thrives in light, drought-resistant, waterloggingresistant, barren, and saline-resistant conditions.The cake meal left after oil extraction can be used as concentrate feed for livestock and poultry.The stems and leaves of C.esculentus can also be used as high-quality fodder, which can be fed directly or made into hay in the sun [1].C.esculentus is a non-food oilseed plant.Its oil has a protein and amino acid-rich composition, with an oil content of approximately 20% [2].Biodiesel produced from the crude oil of C.esculentus seeds exhibits favourable fuel properties [3].Dof (DNA binding with one finger) gene family is a plant-specific class of transcription factors that belong to the single zinc finger protein superfamily [4][5][6].These factors typically consist of 200-400 amino acids and contain oligomerisation sites, nuclear localisation signals, and two functional structures [7,8].The protein has two main regions: the DNA-binding region located at the N-terminus, which is composed of 52 amino acids with a single zinc-finger conserved structural domain rich in cysteine residues, and the transcriptional regulatory region located at the C-terminus, which consists of a single zinc-finger conserved resultant region of a tryptophan [9,10].Dof gene family members recognize either the AAAG sequence-rich or the CTTTT sequence as the core recognition site.This site interacts with DNA or proteins to regulate gene expression and has a dual function [11,12].
In 1993, scientists identified and reported the first Dof transcription factor from maize.Its main function is to participate in the regulation of plant photosynthesis [13].A variety of Dof genes have been identified in plant genomes, with the number of Dof genes varying among different plants.For example, rice has 30 Dof family genes [14], Arabidopsis thaliana has 36 Dof family genes [14], soybean has 78 Dof family genes [15], and potato contains 35 Dof family genes [16].Research has demonstrated that Dof gene family plays a crucial role in various biological processes related to plant growth and development, including plant growth regulation, signal transduction, seed germination, and abiotic stress [4].In Arabidopsis thaliana, various conditions such as salt, drought, high temperature and abscisic acid induced the regulation of AtDof3.3 gene expression, and there was a positive correlation between the high expression level of the AtDof3.3gene and the degree of tolerance to abiotic stress [17].The expression of TaDof2, TaDof3, and TaDof6 genes significantly increased under drought and nitrogen stresses in wheat, suggesting a potential role of TaDof genes in response to abiotic stress [18].VcDof2 and VcDof45 are believed to have significant roles in blueberry flowering and fruit development.Additionally, VcDof1, VcDof11, and VcDof15 were found to respond positively and up-regulate their expression under abiotic stress, indicating potential roles in the defence of blueberries against such stress [19].Tomato's SlCDF3 is highly expressed under the influence of short sunlight, inducing the expression of SlSP5G2 and SlSP5G3.The transcription of SlSP5G is induced under the influence of long sunlight, leading to late flowering in tomato [20].The oil content of cotton seeds grown on land is linked to GhDof1.When a large amount of GhDof1 is expressed, the oil content of the seeds increases while the protein content decreases [21].The expression of Dof proteins in collard green rapeseed is affected by low temperatures.Under cold treatment, the expression of BnCDF1 increases, and the constitutive overexpression of BnCDF1 enhances the frost tolerance of plants [22].Salt stress inhibits the expression of rice OsDOF15 in the root system.On the other hand, overexpression of OsDOF15 limits ethylene synthesis, which results in reduced root sensitivity to salt stress.This suggests that OsDOF15-mediated ethylene biosynthesis may be involved in the inhibition of primary root elongation by salt stress [23].IbDof2, IbDof16, and IbDof36 are up-regulated in response to abiotic stress sources and hormones, and may play a key role in stress tolerance [24].The grape VaDof17 gene overexpression has been found to enhance cold tolerance [25].In chrysanthemum, CmDOF16 is associated with root development, while CmDOF20 and CmDOF21 are significantly more expressed in reproductive organs than in nutrient organs, indicating their crucial role in reproductive development [26].Additionally, CaDof6, CaDof14, CaDof16, and CaDof28 of chilli were found to be highly expressed in the root system [27].The study presented above establishes the basis for identifying and analysing Dof gene family in C.esculentusat a genome-wide level.
Currently, research on cloning and functional studies of Dof proteins is primarily focused on common economic plants such as Arabidopsis thaliana, soybean, maize, rice, and wheat.However, there are few reports on related studies of Dof gene family in Cyperus species, particularly C.esculentus.One study found that CEP2C19 enhances drought tolerance in C.esculentus by regulating ABA sensitivity [28].Scholars combined PacBio High Fidelity (HiFi), Illumina Novaseq, and Hi-C technologies to perform the first high-quality and chromosome-scale genome assembly of C.esculentus [29].The phylogeny of the PP2C gene family in C.esculentus was analyzed through a series of experimental manipulations, including RNA isolation and qRT-PCR, drought and ABA treatments of C.esculentus and Arabidopsis genes.The analyzes concluded that increased overexpression of CePP2C19 affects drought tolerance, particularly in the early stage of seedlings, and significantly reduces ABA sensitivity [28].Scholars have analyzed the physicochemical properties of C.esculentus seeds and found that they contain various substances, such as carbohydrates, starch, and saturated fatty acids [30].In addition, transcriptomics and lipidomics were used by some scholars to track and analyze the key regulators of lipid biosynthesis during C.esculentus tuber development [31].Scholars have studied the CeDGAT2-2 protein of C.esculentus and found that it has high enzyme activity in catalysing TAG formation and strong specificity for oleic acid substrates.
Overexpression of CeDGAT2-2 in C.esculentus tubers enhances the accumulation of oil and oleic acid in the tobacco leaf [32].
C.esculentus is a promising cash crop, both as an important oil plant and as a concentrate feed for livestock.Current research on C.esculentus focuses on its nutrient composition, physicochemical properties, cultivation technology, and medicinal value [33][34][35][36][37]. Zhao et al.his indicates that Dof gene structure is conserved, which is consistent with the gene structure reported in lotsis [29].However, no research has been conducted on the physicochemical properties, amino acid composition, secondary structure, or cis-acting elements of Dof family in C.esculentus.This study utilised bioinformatics methods to identify and analyze the members of the Dof gene family in the entire genome of C.esculentus.Additionally, we performed homology comparisons and constructed a phylogenetic tree with the members of Dof family in the genomes of ten other species, including Arabidopsis thaliana.We propose to explore the relationship and the evolutionary origins of Dof gene family in the genomes of between C.esculentus and other species.These results will provide a reference for future research on the functions of Dof gene family in C.esculentus and the breeding of C.esculentus.

Data acquisition, experimental materials and design
The whole genome sequence, protein sequence, and gene annotation files of C.esculentus were downloaded from China National GeneBank DataBase (CNGBdb)(https:// ftp.cngb.org/ pub/ CNSA/ data5/ CNP00 03839/ CNS06 48185/ CNA00 51961/) [29] The genome sequences, protein sequences and annotation files of Arabidopsis thaliana, Carex littledalei, Rhynchospora breviuscula, Rhynchospora pubera and Rhynchospora tenuis were downloaded from National Genomics Data Center (https:// ngdc.cncb.ac.cn/ gwh/).The protein sequences and gene sequences of Dof gene family of C.esculentus and other species above were extracted from the protein sequences and genome sequences of their species using the TBtools tool after being identified as a Dof gene family by Pfam modelling of Dof gene families and SMART searches for Dof conserved domain, and all the Dof protein sequences and gene sequences were used for subsequent bioinformatics analysis.
C. esculentus seedlings are planted in Key Laboratory of Sichuan Province for Bamboo Pests Control and Resource Development of Leshan Normal University from July 1th to Nov 30th, 2023.Two experimental treatments were conducted on seedlings in laboratory pots, one involving drought stress and the other involving salt stress.The first experimental design is as follows:control groups(CK) were set up, and Cyperus esculentus seedlings were watered normally for 7 d, and the experimental group(T1,T2) were drought-treated with O. latifolia seedlings for 7 d and 14 d.The second experimental design is as follows: The group without NaCl added was designated as the control group (CK), while the groups with 3 g/L, 6 g/L, and 12 g/L NaCl added were designated as treatment groups (T1, T2, T3).The total RNA was extracted from young leaves of the treatment group and the control group.Reverse transcription of purified RNA into cDNA using a reverse transcription kit, and reverse transcribed cDNA was used for qRT-PCR to verify the expression of Dof transcription factor family members in Cyperus esculentus.Plant RNA extraction kit and cDNA reverse transcription kit are purchased from TIANGEN Biotech(Beijing)Co.,Ltd.The qRT-PCR experiments in this study were all completed on fluorescence quantitative PCR instrument (qToWer3 G) of the Analytick Jena AG.The number of replicates of biological samples in each treatment group and control group is 3, and the number of machine replicates on fluorescence quantitative PCR is 3.All CesDof gene primers designed by TBtools Batch q-RT-PCR primer design tool used in the qRT-PCR validation experiment in this study are shown in Supplementary Table 1.The qRT-PCR primers used in this study were synthesized by Sangon Biotech (Shanghai) Co., Ltd on commission.

Identification, chromosomal localization and gene structure analysis of CesDof gene family
We analyzed conserved domains of CesDof proteins by using the online software SMART (http:// smart.emblheide lberg.de/) and identified family genes and simplified selected sequences by using Tbtools software.We carried out chromosomal localisation analysis of the Dof gene family by using MapChart software.The gene structure of CesDof genes was mapped and the exon and intron structure was analyzed using the GSDS 2.0 tool (http:// gsds.cbi.pku.edu.cn/).

Physicochemical properties analysis of CesDof proteins
The molecular weight, isoelectric point, instability index, total average hydrophobicity, and lipolysis coefficient of CesDof proteins were analyzed using the online tool Protparam (http:// web.expasy.org/ protp aram/).

Secondary structure and conserved motif analysis of CesDof proteins
The CesDof proteins' secondary structure was predicted by using the SOPMA tool (https:// npsa-prabi.ibcp.fr/ cgi-bin/ npsa_ autom at.pl? page= npsa_ sopma.html).The MEME online tool (http:// meme-suite.org/ tools/ meme) was used to analyze the CesDof proteins' conserved motifs, with the number of predictions set to 10 in the parameters and all other parameters set to default.

Evolutionary tree analysis of CesDof proteins
Sequence comparison of CesDof protein sequences was performed using Clustalx.The phylogenetic tree of Ces-Dof proteins was constructed using MEGA7 software with Neighbor-Joining (NJ) method and the calibration parameter BootStrap repeated 1000 times.The same method was used to construct the evolutionary tree of C.esculentus and Arabidopsis thaliana, as well as the evolutionary tree of C.esculentus and Salvia officinalis.Phylogenetic trees of Dof protein sequences from six species, including C.esculentus, Arabidopsis thaliana, Rhynchospora tenuis, Rhynchospora pubera and Rhynchospora breviuscula, were constructed using Maximum Likelihood (ML).The calibration parameters were repeated 1000 times, and bootstrap was repeated 1000 times.

Analysis of cis-acting elements of CesDof genes
The promoter sequences of each member of the Dof gene family were selected from C.esculentus genome database.These sequences consist of 2000 bp upstream of the ATG.They were imported into text and collated into a fasta file.The file was then submitted to the PlantCARE website for promoter cis-acting element analysis.The sequences were filtered and sorted based on the results.Finally, they were visualised and analyzed using the 'Simple Bio Sequence Viewer' module in Tbtools.

Transcription factor binding sites analysis of CesDof genes
The promoter sequences of each member of Dof gene family were obtained from C.esculentus genome database.A 2000 bp sequence upstream of the ATG start codon was used.The sequences were organized into a fasta file and submitted to PLANTREGMAP website for transcription factor binding site analysis.The data was sorted, filtered, and analyzed using the 'Simple Bio Sequence Viewer' in TBtools, following the analysis structure.

Codon preference of CesDof genes
Relative synonymous codon usage (RSCU), codon adaptation index (CAI), GC content of synonymous codons (GC), Effective Number of Codon (ENC/Nc), Codon Bias Index (CBI), frequency of optical codons (Fop), and frequency of optimal codon usage (Fop) of CesDof genes were obtained by Codon W analysis.

Collinearity and KaKs analysis of CesDof genes
Using default parameters, MCSCANX software was used to analyze the large segment tandem repeat events in CesDof gene family members.Based on the specific chromosome location information of the family members, TBtools software was used to analyze the collinearity of Dof genes in C.esculentus genome.The collinearity analysis between C.esculentus and Arabidopsis thaliana, Carex littledalei, Rhynchospora tenuis, Rhynchospora pubera, and Rhynchospora breviuscula.The simple Ka/Ks calculator of TBtools was used to analyze the KaKs of CesDof with collinearity.

Analysis of SSR loci and microRNA target prediction for CesDof genes.
The SSR loci of CesDof genes were identified by using MISA-web (https:// webbl ast.ipk-gater sleben.de/ misa/) with modification parameters described by Gao et al. (2011).psRNATarget (https:// www.zhaol ab.org/ psRNA Target/; Dai et al., 2018) with an expected value of 3 and default parameters, was predict to potential CesDof gene miRNAs from all miRNAs in the database.

Protein-protein interactions analysis of CesDof proteins
The STRING interactive database was used to construct the interaction network between CesDof proteins.Arabidopsis thaliana was selected as the model plant to construct the protein interaction network diagram.The predicted network interactions were displayed by Cytoscape software.
Gene expression analysis of CesDof gene family.
The RNA extracted from all leaf samples in the two experimental groups and the control group under drought stress and salt stress conditions was reverse transcribed into cDNA and used for qRT-PCR verification experiments.The data obtained from the qRT-PCR experiments were statistically analyzed, and the gene expression data of CesDof gene family under drought stress and salt stress conditions were displayed as a histogram using the online analysis platform omicshare Tools (https:// www.omics hare.com/ tools/ Home/ Soft/ histo gram).The expression level changes of CesDof gene family under drought stress and salt stress conditions were analyzed.
The gene structure diagram of CesDof drawn by using the GSDS2.0tool reveals that there are differences in sequence length and coding sequence length among the 29 CesDof gene sequences, with sequence lengths ranging from 400 to 3800 bp and coding sequence lengths ranging from 10 to 1000 bp.CesDof08, CesDof09, CesDof10, CesDof14, CesDof16, CesDof18, CesDof19, CesDof20, Ces-Dof22, CesDof23, CesDof24, CesDof26 and CesDof27 have one intron and two exons, while the other CesDof gene sequences contain only one exon.CesDof15 has 2 introns and 3 exons (Fig. 2).

Physicochemical properties analysis of CesDof proteins
The physicochemical properties of CesDof proteins were analyzed.CesDof01 was found to have the highest number of amino acids, while CesDof25 had the lowest with 124 amino acids.The overall amino acid content of Ces-Dof protein ranged from 124 to 512 amino acids, with an average of 286.The molecular weight of CesDof proteins ranged from 13.83 to 51.73 kD.The isoelectric point (pI) values ranged from 4.71 to 9.84.However, only 7 CesDof members had a theoretical isoelectric point less than 7, while the rest had a theoretical isoelectric point greater than 7. Therefore, most of CesDof members were basic proteins.Comparison of the instability index values showed that all CesDof sequences were predicted to be unstable proteins, except for CesDof09 and CesDof25 are stable proteins, which had instability index values below 40.All other CesDof sequences had instability index values greater than 40 and were predicted to be unstable proteins.The aliphatic index content of CesDof proteins ranged from 35.76 to 75.39, indicating a wide variation in thermal stability among this family of proteins.According to the prediction of protein hydrophilicity/hydrophobicity, all 29 CesDof members were found to be hydrophilic proteins, with negative GEAVY values.The highest hydrophilicity value (-0.344) was observed in CesDof11, while the lowest (-1.212) was observed in CesDof27.6 CesDof proteins had a negative charge as the total number of negatively charged residues was greater than the total number of positively charged residues.The remaining 23 CesDof members had a positive charge as the total number of positively charged residues was greater than the total number of negatively charged residues (Table 1).

Signal peptide prediction and subcellular localization results of CesDof proteins
All 29 Dof proteins of C.esculentus were predicted to lack signal peptides, indicating that they are non-secretory proteins.Additionally, the subcellular localization prediction showed that all 29 CesDof members were located to the nucleus.These results suggest that the C.esculentus Dof transcription factors primarily function in the nucleus (Table 1).

Transmembrane structure, hydrophilicity and phosphorylation site prediction of CesDof proteins
The transmembrane structure prediction for the Ces-Dof gene family in C.esculentus revealed that none of 29 CesDof proteins members exhibited transmembrane phenomena.Therefore, it was inferred that this family of proteins are non-transmembrane proteins.
The hydrophilicity/hydrophobicity analysis revealed that CesDof protein members had hydrophobicity values ranging from 0.833 to 2.767 and hydrophilicity values ranging from -4.056 to -2.311.The most hydrophobic member was CesDof01 with a value of 2.767, while the most hydrophilic member had a value of -4.056.The amino acid scores were inversely proportional to the hydrophilicity of the members, with lower scores indicating higher hydrophilicity and higher scores indicating higher hydrophobicity.CesDof01 has a hydrophobic alanine at position 112, and hydrophilic arginine and glutamic acid at positions 35, 36, and 37. Overall, CesDof proteins are hydrophilic, with more numerous and denser hydrophilic peaks than hydrophobic peaks (Table 2).
Phosphorylation site analysis of CesDof proteins revealed a total of 1141 serine (Ser) phosphorylation sites, 433 threonine (Thr) phosphorylation sites, and 147 tyrosine (Tyr) phosphorylation sites in the 29 CesDof members.Among CesDof protein members, CesDof01 has the highest number of serine (Ser) phosphorylation sites, with a total of 78.The most probable phosphorylation site is serine (Ser) at position 206, with a value of 0.998.CesDof01 also has the highest number of threonine (Thr) phosphorylation sites, with a total of 34.The most probable phosphorylation site is threonine (Thr) at position 420, with a value of 0. 875.CesDof21 has the highest number of tyrosine (Tyr) phosphorylation sites, with a total of 9.The most probable phosphorylation site is tyrosine (Thy) at position 193, with a value of 0.985.CesDof01 has the highest total number of phosphorylation sites, with a total of 78.CesDof22 and CesDof24 have the lowest total number of phosphorylation sites, with only two each.Given that the serine content is highest in this family, it is likely that the proteins primarily carry out their functions through phosphorylation at the serine (Ser) site (Table 3).

Conserved structure of CesDof proteins
The conserved structure of CesDof proteins were analyzed using the online tool MEME, resulting in the identification of 10 independent conserved motifs.The lengths of these motifs ranged from 11 to 41 amino acids(Supplementary Fig. 2A).Of the 10 conserved motifs, motif1 is a single zinc finger structure (C2-C2) and is present in 29 family members.This suggests that it is a core component of the Dof proteins of C.esculentus.It is hypothesized that the different numbers and types of motifs in different Dof proteins may be due to their varying functions in the organism.Out of the 29 CesDof members, each one contains at least one conserved motif, with a maximum of eight.20 CesDof members all contained motif1, while 2 CesDof members contained other motifs, excluding motif2 (Supplementary Fig. 2B).

Evolutionary tree analysis of CesDof proteins
The 29 CesDof proteins can be divided into four distinct groups (labelled Group1, Group2, Group3 and Group4) based on their degree of aggregation in the tree.Group4 has the highest number of CesDof protein members, with 10 CesDof protein members, accounting for 34.48% of the total.Meanwhile, Group1 and Group3 have the lowest number of CesDof protein members, each with 6 Ces-Dof protein members, accounting for 20.69% of the total.Group1 is considered the most primitive evolutionary group within the family, consisting of 6 CesDof protein members.Among them, CesDdof29 is the most primitive in terms of evolution, while CesDof08 is the fastest evolving sequence.Group4, on the other hand, is the fastest evolving group with 10 CesDof protein members.Among them, CesDof07 is the most primitive in terms of evolution, while CesDof20 is the fastest evolving (Fig. 3).The phylogenetic evolutionary tree of Dof gene family of C.esculentus and Carex littledalei reveals that the Dof proteins of both species can be classified into nine groups (Group1 to Group9).Group1 was the least evolved, consisting of 4 members of C.esculentus and 4 members of Carex littledalei.Among them, CesDof24 and CliDof21 were the most primitive, while CliDof13 and CliDof14 were the fastest to evolve.Group9, on the other hand, was the most evolved, comprising 5 members of C.esculentus and 3 members of Carex littledalei.CesDof29 was the most primitive in Group9.Group 2 includes one member of C.esculentus and one member of Carex littledalei.Group 3 includes nine members of C.esculentus and eight members of Carex littledalei.Groups 4, 5, and 7 each contain one member of C.esculentus and one member of Carex littledalei.Group 6 includes one member of C.esculentus and one member of Carex littledalei.Group 7 also contains one member of C.esculentus and one member of Carex littledalei.Group 6 comprises five members of the C.esculentus family and three members of the Carex littledalei family.Group 8 consists of three members of the C.esculentus family and three members of the Carex littledalei family (Fig. 4A).
The phylogenetic evolutionary tree of Dof gene family in C.esculentus and Arabidopsis thaliana reveals that their phylogenetic relationships can be broadly classified into 11 groups (labelled sequentially as Group1, Group2, Group3, Group4, Group5, Group6, Group7, Group8, Group9, Group10, Group11).Group11 was the least evolved group, consisting of only one member from C.esculentus and one member from Arabidopsis thaliana.On the other hand, Group10 was the fastest evolving group, comprising of eight members from Arabidopsis thaliana and six members from C.esculentus.In Group 10, CesDof23 is the most primitively evolved, while Ath-Dof22 and AthDof44 are the fastest evolved.Group 1   Group 5, Group 6, Group 7, Group 8, Group 9, Group 10, Group 11, Group 12 in sequence).Overall, the differentiation of Group 1 is relatively primitive, with one member from C.esculentus and one member from Rhynchopora breviscula.The Group12 group has the fastest evolution, with 5 members of C.esculentus and 3 members of Rhynchopora breviscula.Among them, CesDof29 has the most primitive evolution, while CesDof06 and RbrDof12 have the fastest evolution.Group 2, Group 3, Group 4, Group 6, and Group 10 all contain one C.esculentus and one Rhynchopora breviuscula member.Group5 contains 2 members of C.esculentus.There are 5 members of C.esculentus and 3 members of Rhynchopora breviscula in Group 7.There are 4 members of C.esculentus and 4 members of Rhynchopora breviscula in Group 8.There are 4 members of C.esculentus and 2 members of Rhynchopora breviscula in Group 9. In Group 11, there are three members of C.esculentus and Rhynchopora breviuscula, respectively (Fig. 4D).
According to the phylogenetic tree of Dof gene family in C.esculentus and Rhynchopora tenuis, the phylogenetic relationship of Dof proteins in C.esculentus and Rhynchopora tenuis can be roughly divided into 13 groups (labeled as Group 1, Group 2, Group 3, Group 4, Group 5, Group 6, Group 7, Group 8, Group 9, Group 10, Group 11, Group 12, Group 13).Overall, Group12 evolved the most primitive, including two members, Ces-Dof24 and RteDof05.Group 13 has the fastest evolution, including 7 members of C.esculentus and 2 members of Rhynchopora tenuis.Among Group 13, CesDof19 and CesDof20 have the most primitive evolution, while Ces-Dof28 and RteDof17 have the fastest evolution.Group 1, Group 2, Group 3, Group 5, Group 7, Group 11, and Group 13 each contain one C.esculentus member and one Rhynchopora tenuis member.Group 4 includes 2 members of C.esculentus.Group 6 includes 5 members of C.esculentus and 3 members of Rhynchopora tenuis.Group8 contains 3 members of C.esculentus and Rhynchopora tenuis, respectively.Group9 contains 4 members of C.esculentus and Rhynchopora tenuis, respectively.Group 10 only contains one C.esculentus member (Fig. 4E).
According to the phylogenetic tree of Dof gene family between C.esculentus and five other species, the phylogenetic relationship of Dof proteins in C.esculentus and the other five species can be roughly divided into 10 groups (labeled as Group 1, Group 2, Group 3, Group 4, Group 5, Group 6, Group 7, Group 8, Group 9, Group 10).Overall, Group1 evolved the most primitive, consisting of three Arabidopsis members, one C.esculentus member, four Rhynchopora pubera members, one Rhynchopora breviscula member, and one Rhynchopora tenuis member.Group 5).

Transcription factor binding sites of CesDof genes
Tbtools was used to predict the transcription factor binding sites of CesDof gene family.The results showed that there were C2H2, ERF, MYB, LBD, BBR-BPC, MYB_ related, Dof, B3, CAMTA, NAC, TCP, WRKY, WOX, LFY, VOZ, ARR-B transcription factor binding sites in CesDof genes.Dof, ERF, MYB, BBR-BPC, MIKC_MADS in CesDof genes was the most binding sites for these five transcription factors.Among them, in CesDof transcription factors are involved in various biological processes during plant growth and development, regulating light response and carbon and nitrogen metabolism, seed development and

Codon preference analysis of CesDof genes
The codon preference analysis of different codons for each amino acid (RSCU), codon adaptation index (CAI), synonymous codon GC content (GC), and 3rd base content (GC3s, T3s, C3s, A3s, G3s), as well as the effective number of codons (ENC/Nc) and codon bias index (CBI) were obtained using CondonW analysis.The results show that CAI ranges from 0 to 1, with lower codon preference degrees resulting in CAI values closer to 0. The codon preference ranges from a minimum of 0.135 for CesDof11 to a maximum of 0.275 for CesDof29.The Nc value ranges from 41.31 (CesDof25) to 57.81 (CesDof26), with larger values indicating weaker codon preference and smaller values indicating stronger codon preference.The GC content, which is the total amount of GC in the codon, ranged from 0.671 (CesDof15) to 0.362 (CesDof25), with an average content of 0.495.The GC3s content ranged from 0.763 (CesDof15) to 0.331 (CesDof25), with an average content of 0.496.The codon 3rd base contents were T (0.329) > C (0.326) > G (0.287) > A (0.286).The content of T/A was higher than the content of G/C, indicating that the CesDof gene preferred to end in T/A.The CBI index was used to assess the potential expression of exogenous genes in the target hosts.A value of 1 indicates that all codons are optimally used, while a value of 0 indicates that all codons are used randomly.The range of CBI is from -0.117 (CesDof25) to 0.304 (CesDof26).The Fop value ranges from 0 to 1, with 1 indicating the use of only optimal codons and 0 indicating the absence of optimal codons.The content ranges from 0.332 (CesDof25) to 0.598 (CesDof25)(Supplementary Table 3).
The RSCU values of the codons in the CesDof gene family of C.esculentus analyzed using CodonW software.The results showed that 30 codons had RSCU values greater than 1, with only 7 of them ending in G/C.AGA was the most strongly preferred codon for C.esculentus, while AUG, AUA, GGC, and UGG had no codon preference (RSCU = 1).Additionally, 30 codons, including CUC, CUA, and CUG, had very low codon preference (RSCU < 1) (Supplementary Table 4).
The ratio of nonsynonymous substitutions (Ka) to synonymous substitutions (Ks) can indicate whether selection pressure acted on the protein-coding gene in the plant.The Ka/Ks ratios for the CesDof replication events ranged from 0.21 (CesDof22/CesDof29) to 0.35 (Ces-Dof24/CesDof12), all less than 1, suggesting that all Ces-Dof replication events underwent purifying selection.CesDof is evolutionarily conserved (Table 4)., and rna21024, respectively.These genes belong to a one-to-one collinearity relationship (Fig. 9A).
Figure 10 shows the collinearity maps constructed of the same family between C.esculentus, Arabidopsis thaliana and four other species.The following collinearity relationships were obtained for the six species: 129 pairs of homologous genes between C.esculentus and Rhynchospora pubera, 114 genes in Rhynchospora pubera were in collinearity with Rhynchospora breviuscula, and 31 genes in Rhynchospora breviuscula are collinearity with Carex littledalei genes.There are 29 pairs of homologous gene pairs between Carex littledalei and Rhynchospora tenuis, as well as 18 pairs of collinearity gene pairs between Rhynchospora tenuis and Arabidopsis thaliana.These findings suggest that all six species share a common ancestor, with greater collinearity observed between plants of the same family (Fig. 10).

Analysis of SSR loci in CesDof genes
The SSR loci analysis shows that the CesDof gene or promoter contained a total of 22 SSR sites, which were classified into 5 types: p1, p2, p3, p6, and c.Among them, p1 had 10 types, p2 had 4 types, and both p3 and p6 had only 1 type each, namely CesDof10 and CesDof13, respectively.Additionally, there were 6 types of c.The SSR length of CesDof22 was the longest at 103, while CesDof16 had the shortest SSR length at 10(Supplementary Table 5).

MiRNAs prediction of CesDof genes
These miRNAs of Dof genes have the function of regulating gene expression, therefore we predicted candidate miRNAs targeting the CesDof genes.It is expected that 144 miRNAs will target 26 CesDof genes.Among them, ath-miR5021 is predicted to target the most Ces-Dof genes, with a total of 7, namely CesDof08, CesDof09, CesDof10, CesDof15, CesDof14, CesDof19 and CesDof26; Next is ath-miR5014a-3p, which is predicted to target 5 CesDof genes, namely CesDof04, CesDof08, CesDof10, CesDof15, CesDof27.Other miRNAs are predicted to target one or different types of CesDof genes.Predicting 22 different miRNAs as target genes for the CesDof08 gene; Next is the CesDof15 gene, which has 19 different target genes for miRNAs.CesDof04 and CesDof21 genes have the least number, with only one target gene for different miRNAs, namely ath-miR5014a-3p and ath-miR1888a(Supplementary Table 6).

Protein-protein interactions of CesDof proteins
Protein-protein interaction analysis showed protein network of CesDof protein interactions was constructed by using the STRING online database.The results indicate an interaction relationship between the proteins of GI, ADO3, TDR, GA3OX1, LBD4, CesDof17, CesDof27, CesDof23, CesDof15, CesDof07, CesDof34, CesDof18, CesDof20, CesDof26, CesDof25, CesDof12, CesDof03, Ces-Dof29, CesDof19 and CesDof21.The diagram shows the interaction network of functional genes, with lines connecting them and the thickness of the lines indicating the strength of the interaction.The size and colour depth of the nodes indicate that CesDof12, CesDof03, CesDof29, CesDof19, GI and ADO3 are the core proteins.CesDof12 interacts with 13 proteins, while CesDof03 and Ces-Dof29 interact with 12 proteins each.CesDof19, GI and ADO3 have 10, 7, and 7 interacting proteins, respectively (Fig. 11).

Gene expression analysis of CesDof gene family under drought stress and salt stress conditions by qRT-PCR
Through qRT-PCR experiments on CesDof gene family in the leaves of C.esculentus seedlings after 7 days and 14 days of drought treatment, the results showed that compared with the control group (CK), gene expression level of 11 CesDof genes (CesDof01, CesDof03, CesDof04, CesDof07, CesDof10, CesDof11, CesDof12, CesDof13, CesDof17, CesDof21, CesDof28) in C.esculentus in the drought treatment group (T) were higher than those in the control group.The expression levels of 9 CesDof genes in the drought treatment group (T), including CesDof02, CesDof05, CesDof06, CesDof08, CesDof09, CesDof14, Ces-Dof15, CesDof22 and CesDof23 were lower than those of the control group.The remaining 9 CesDof genes in this gene family showed little change in expression content after drought stress treatment.These results suggest that most CesDof genes are very responsive to drought stress, indicating that most CesDof genes may be involved in drought stress responses (Fig. 12).
The qRT-PCR analysis of the salt stress treatment experiment showed that compared with the control group, the gene expression levels of 11 CesDof genes increased with increasing NaCl concentration, namely CesDof01, CesDof03, CesDof04, CesDof07, CesDof10, CesDof11, CesDof12, CesDof13, CesDof17, CesDof21, Ces-Dof28.The expression levels of 9 CesDof genes decreased, namely CesDof02, CesDof05, CesDof06, CesDof08, Ces-Dof09, CesDof14, CesDof15, CesDof22 and CesDof23, and only the remaining 9 CesDof genes showed little change in expression levels.These results indicate that most Ces-Dof genes have a relatively large response to salt stress, and suggest that CesDof gene family may be involved in their salt stress response (Fig. 13).We were pleasantly surprised to find that the mechanisms of CesDof gene family in response to drought and salt stress are similar, and the change trends of gene expression levels of many CesDof genes in response to drought and salt stress are consistent.

Discussion
Dof gene family is a plant-specific transcription factor that plays a crucial role in various physiological processes, including plant growth and development.Currently, Dof gene family of plant genomes has been identified in various plants, such as rice, wheat, maize, and tobacco.However, there are no relevant reports on the whole genome of the Dof gene family in the perennial  which is a similar number to those found in rice (30), tomato (34) and lotus (29).We analyzed their physicochemical properties, chromosomal localization, and secondary and tertiary structures.The chromosome localization was also examined.The Dof gene family of C.esculentus was analyzed for various aspects including secondary and tertiary structures, phosphorylation sites, hydrophilicity and hydrophobicity, conserved motifs, cisacting elements, transcription factor binding sites, phylogenetic tree analysis, collinearity analysis, SSR analysis, miRNA prediction, and protein interaction.
Analysis of structural domains and motifs of 29 Dof gene families in C.esculentus revealed that they all contain complete C2-C2 single finger zinc structures, which is consistent with the studies of Zou [4], Li [9].Different gene families have unique motifs, and CesDof has a common motif in C.esculentus, indicating that the conserved motif plays an important role.In addition, the diversity of motifs between different CesDof may be related to their complex functions.
Within the 29 family members, the majority of C.esculentus Dof gene family either lack introns or have only one.This indicates that the Dof gene structure is conserved, which is consistent with the gene structure reported in lotus [38], wheat [12], rice [39], and tomato [40].
The isoelectric point (pI) value of C.esculentus DOF family ranges from 4.71 to 9.84, according to the physicochemical properties of the C.esculentus DOF protein.This is similar to the results predicted by scientists who identified the tobacco Dof gene in the common tobacco K326 genome [41] and DOF gene in the annual alfalfa identification [42].Most members are basic and unstable proteins, which is consistent with the research structure of Sunshouru and other scholars on American pumpkin [43].All 29 members were hydrophilic, non-secretory proteins, and located in the nucleus, indicating that the DOF protein plays a regulatory role in plant growth and development.This positioning is consistent with the prediction result of Rose [44].
The analysis of amino acid transmembrane structure, hydrophobicity, and phosphorylation sites of DOF family members indicates that the members of DOF family in C.esculentus are hydrophilic proteins and lack transmembrane domains.Their protein functions are mainly realized through phosphorylation at serine sites.These results are consistent with those predicted by scholars in Brassica napus [45].The secondary structure prediction shows that C.esculentus DOF family members are primarily composed of random coils, α-helix, extended chain structures, and relatively small amounts of β-turns, with the lowest occurrence of β-turns.The description of β-turns and extension chains is scattered throughout the entire amino acid.The online tool MEME was used to This motif is present in all 29 family members, which is consistent with research on the transcription factor of linseed mustard Dof [46].These results suggest that Dof proteins with the same conserved motifs in the same group may have the same function.
By analyzing the promoter region of 2000 bp upstream of CesDof sequence, it was found that they may play a role in hormone response, light response, growth and development, and the light response element is one of the typical characteristic elements.Previous studies have shown that in Arabidopsis, if the DOF of OBP strain is highly expressed, the light response elements will change, and then affect the growth of Arabidopsis [47].In Phyllostachys edulis, PheDof12-1 can regulate photoperiod related regulatory factors when put PheDof12-1 gene into different environments, such as cold, drought, salt and gibberellin (GA3).Through ectopic expression in Arabidopsis, it was found that the overexpressed homozygous PheDof12-1 of transgenic Arabidopsis showed early flowering under long day (LD) conditions, combined with the promoter sequence of PheCOL4, and had a strong circadian pattern, These results indicate that DOF transcription factor is involved in regulating the flowering time of Phyllostachys heterocycla [48].This suggests that the members of the DOF family may be closely related to the light regulatory elements.In addition, DOF also affects the regulation of plant growth and development by regulating the expression of a variety of hormones.For example, the DOF transcription factor ntbbf1 (rolB domain B factor 1) in Nicotiana tabacum binds to the acttta region of the gene rolB promoter, thereby regulating the tissuespecific expression of rolB gene and the growth factor induced expression [49], promoting root growth [50].The analysis of cis element of promoter will lay a foundation for further study of the potential function of CesDof involved in light signal and hormone related pathways.The Dof genes may play an important role in plant growth, light response, hormone response and stress response.This is consistent with the previous studies that the Dof gene family responds to light and participates in metabolism.
Further study of transcription binding factors identified 44 types in 29 CesDof, including Dof, ERF, MYB, BBR-BPC, MIKC_MADS.etc.This suggests that various transcription binding factors play a crucial role in the growth, development, reproduction, and other physiological processes of C.esculentus, and they must coordinate with each other.Research has shown that the DOF protein is a multifunctional transcription factor that affects various aspects of plant growth and development, including light response, tissue differentiation, seed development or germination, and plant hormone signal transduction [51].For instance, when rice highly expresses OsDof04, it can respond to light and affect flowering time depending on the duration of light exposure [52].Apple MdDof54 regulates the expression of drought response genes, root development, and photosynthesis, affecting drought resistance.It also shows a positive correlation [53].Overexpression of PhDof28 in Petunia hybrida promotes the accumulation of IAA, resulting in petal elongation [54].
The codon preference of 29 C.esculentus DOF genes was investigated.It was found that the gene preference of C.esculentus ended with T/A, and AGA (RSCU = 1.48) was the 1 synonymous codon with the strongest preference for C.esculentus.AUG, AUA, GGC and UGG had no codon preference (RSCU = 1), while others were less than 1.The ENC ranged from 41.31 to 57.81, with an average of 52.93.The CAI value ranged from 0.135 to 0.275.This is similar to the research findings of the strawberry DHAR gene preference [55] and the Siberian apricot pssoc1-like gene codon preference [56].These results indicate that the C.esculentus gene has a weak codon preference and a low transcription expression level.
MIRNAs are small endogenous RNAs that exist widely in plants and function in post-transcriptional regulation of gene expression.They play an important role in plant growth, development, and abiotic stress response [57].In bananas, 30 miRNAs are estimated to target 12 mafad members, with ac-mir156 and mac-mir164 targeting the largest number of genes [58].19 miRNAs are predicted to target twenty ahfads in peanuts, including four FAD3s, four FAD2s, two FAD7s, one FAD8, and eight SLDS [59].The study discovered that 144 miRNAs were expected to target 29 CesDof genes, with ath-mir5021 targeting seven different CesDof genes.It is possible that these miRNAs directly regulate the expression of these CesDof genes, but this requires confirmation in future studies.
The phylogenetic tree for the Dof genes of C.esculentus and other plants was constructed by the author.The results indicate that the 29 Dof genes of C.esculentus were divided into four categories based on their phylogenetic relationship and sequence similarity.This finding is consistent with previous research on Dof gene family of Arabidopsis, rice and wheat [8,[60][61][62].Most of the CesDof genes within the same subfamily exhibit similar motifs, suggesting that these conserved motifs may be crucial for group or subgroup-specific functions.The distribution of motifs among different subfamilies suggests the complexity of protein function in the DOF gene family of C.esculentus.However, the number of introns in CesDof genes was mostly between 0 and 2, indicating evolutionary conservation.There were no significant differences between the exons and introns of CesDof gene family members.The phylogenetic tree of 29 CesDof genes and AthDof genes was constructed by the author, which were roughly divided into five groups.CesDof genes were significantly reduced in the process of plant evolution, leaving only AthDof genes in group1 and only CesDof genes in Group9.Therefore, it is speculated that there are differences in the evolution of DOF genes in different species.The phylogenetic analysis of Dof gene family in C.esculentus, Cyperaceae, Arabidopsis thalianaand other species revealed that Dof gene family tree in C.esculentus and six other plants was divided into ten groups, namely group 1 to group 10.CesDof protein members were primarily distributed in Group 4, 7, and 10, with some presence in Group 3 and 9. Group 6 and 8 did not contain any CesDof protein members, indicating that the homologous genes of CesDof in these two groups may have been lost during the evolutionary process.
To investigate the origin and evolution of Dof gene of C.esculentus, we conducted a collinearity analysis of the Dof gene families of C.esculentus, Arabidopsis thaliana, and other Cyperaceae plants.The analysis revealed three pairs of homologous genes among the 29 CesDof genes.Furthermore, most of CesDof genes did not have any homologous gene members on chromosomes.The uneven distribution of CesDof gene family during chromosome evolution may explain this phenomenon.Collinearity analysis revealed that 15 out of 29 CesDof genes were collinear with Arabidopsis thaliana.Both CesDof18 and CesDof14 had three homologous genes, indicating a relatively close genetic relationship between Arabidopsis thaliana and C.esculentus.The Dof gene has been conserved throughout the process of plant evolution, even among different species.In C.esculentus, many genes in Arabidopsis have more than two homologous genes, indicating a shared origin between the two.Thirty-one of Carex littledalei genes were collinear with CesDof, and 79% of CesDof had 1-2 homologous genes in Carex littledalei.Twenty-eight of the genes were collinear with Rhynchospora tenuis, representing 72% of the total.Additionally, 33 genes were collinear with Rhynchospora breviuscula, accounting for 76% of the total.There was a colinearity relationship between 129 Rhynchospora pubera genes and CesDof genes.Further statistical analysis of the homologues of Dof genes in Rhynchospora pubera in C.esculentus revealed that 83% of CesDof genes had homologues in Rhynchospora pubera.Among them, CesDof19 had six homologous genes in Rhynchospora pubera.This suggests that the amplification of Dof gene family may have occurred prior to the differentiation of To identify the selection pressure of the CesDof repeat gene in the evolution process of C.esculentus, three Ces-Dof repeat events were analyzed by Ka/Ks.If the Ka/Ks ratio is greater than 1, it is considered that CesDof gene has undergone a positive selection effect in the evolutionary process, accelerating its evolution.When the Ka/Ks ratio is equal to 1, neutral selection is considered to be acting on the CesDof gene, meaning that natural selection does not affect its evolution.If the Ka/Ks ratio is less than 1, it is considered that the CesDof gene has undergone purifying selection [63].It was found that the Ka/Ks values of the three CesDof repeat events ranged from 0.21 (CesDof22/CesDof29) to 0.35 (CesDof24/CesDof12), and the ratio was less than 1.Therefore, the CesDof repeat gene was selected during the process of evolution.
Dof proteins have been shown to interact with other proteins [64,65].Interacting protein prediction analyzes indicate that some of C. esculentus Dof family proteins have close interactions with each other.The results indicate that 15 Dof proteins interact with Arabidopsis GI protein, ADO3 protein, TDR protein, GA3OX1 protein and LBD4 protein.Cesdof12 and CesDof29 are homologous to zinc finger proteins Dof2.5 and Dof3.7 of Arabidopsis, respectively.They play a role in maternal control of seed germination and regulate transcription by binding to the common core sequence of 5'-AA[Ag]G-3' .Ces-dof03 is a homologous protein to Arabidopsis zinc finger protein Dof5.6.It specifically binds to the 5'-AA[Ag]G-3' consensus core sequence, promotes the radial growth of protophloem sieve elements, and participates in the formation of interfascicular cambium and the regulation of vascular tissue development, particularly in the early stage of inflorescence stem development.Ces-dof19 and the zinc finger protein Dof5.1 of Arabidopsis thaliana are homologous and can specifically bind to the 5'-AA[AG]G-3' consensus core sequence.They also bind to the 5'-TAA AGT -3' motif in the Rev promoter to trigger its transcription, thereby regulating paraxial polarity and auxin transport, including the expression of IAA6 and IAA19 genes.Gigantea (GI) is a plant-specific circadian clock control gene that interacts with CONSTANS (CO) to regulate the long-day flowering pathway.It primarily regulates photoperiodic flowering and the circadian rhythm of plants [66].This protein has a pleiotropic function in the entire developmental stage, from seed germination to flowering time control [67], and also participates in red light signals and controls circadian clock function.The SCF (ADO3) E3 ubiquitin ligase complex regulates circadian rhythm and has diverse roles.Some core proteins are involved in seed germination, others in circadian rhythm and photoperiod regulation, and some in the radial growth of protophloem sieve elements.These proteins form complexes with DOF and other types of proteins through direct protein-protein interactions, cooperating to regulate the growth of C.esculentus.

Conclusion
In this study, we have identified 29 CesDof genes in C.esculentus genome.They are located in the nucleus and have a range of 124 to 512 amino acids, with most being basic proteins.Their secondary structure is mainly random coil.The promoter sequence of CesDof genes contains cisacting elements that respond to light, drought, hormones, low temperature, and circadian rhythm.The CesDof genes' codon preference ends in T/A.C.esculentus had three pairs of collinear CesDof genes.Additionally, there were 15 pairs of collinear genes shared between C.esculentus and Arabidopsis thaliana.The genetic relationship between C.esculentus and Rhynchospora pubera was found to be the closest.Phylogenetic tree analysis revealed that the 29 CesDof genes of C.esculentus can be classified into 4 subgroups.Additionally, 144 miRNAs were predicted to target these CesDof genes.Furthermore, protein interaction analysis indicated that 15 Dof proteins in C.esculentus have interactions.Most CesDof genes were involved in drought stress and salt stress responses, and the gene expression trends under drought stress and salt stress conditions were consistent These results lay a theoretical foundation for further studying the molecular functions of Dof gene family in C.esculentus and its molecular mechanisms in regulating the life activities of C.esculentus.

Fig. 1
Fig. 1 Chromosome mapping of Dof gene family in C.esculentus

Fig. 2
Fig. 2 Gene structure of Dof genefamily in C.esculentus contains three Arabidopsis members, Group 2 contains six C.esculentus members and eleven Arabidopsis members, Group 3 contains two C.esculentus members, and Group 4 contains six Arabidopsis members and one C.esculentus member.Group 5 comprises 7 members of Arabidopsis and 5 members of C.esculentus.Group 6 comprises 2 members of C.esculentus and 4 members of Arabidopsis.Group 7 comprises 6 members of Arabidopsis and 3 members of C.esculentus.Group 8 comprises 1 member of C.esculentus and 1 member of Arabidopsis.Group 9 comprises 2 members of C.esculentus exclusively (Fig.4B).According to the phylogenetic tree of Dof gene family in C.esculentus and Rhynchopora pubera, the phylogenetic relationship of Dof proteins in C.esculentus and Rhynchopora pubera can be roughly divided into 13 groups (labeled as Group 1, Group 2, Group 3, Group 4, Group 5, Group 6, Group 7, Group 8, Group 9, Group 10, Group 11, Group 12, Group 13).Overall, Group1 evolved the most primitive, including 12 Rhynchopora pubera members and 3 C.esculentus members; Group 14 has evolved the fastest, including 3 members of C.esculentus and 11 members of Rhynchopora pubera.Group 2, Group 6, Group 10, and Group 13 each contain 4 Rhynchopora pubera members and 1 C.esculentus member.Group3 contains 2 members of C.esculentus.Group4 contains 4 Rhynchopora pubera members and 1 C.esculentus member.Group 5 contains 2 members of C.esculentus and 2 members of Rhynchopora pubera, respectively.Group 7 contains 15 Rhynchopora pubera members and 5 C.esculentus members.Group8 contains 7 Rhynchopora pubera members and 2 C.esculentus members.Group9 contains 8 Rhynchopora pubera members and 2 C.esculentus members.Group 11 contains 4 Rhynchopora pubera members and 2 C.esculentus members.Group 12 contains 5 Rhynchopora pubera members and 3 C.esculentus members (Fig.4C).According to the phylogenetic tree of Dof gene family in C.esculentus and Rhynchopora breviscula, the phylogenetic relationship of Dof proteins in C.esculentus and Rhynchopora breviscula can be roughly divided into 12 groups (labeled as Group 1, Group 2, Group 3, Group 4,

Fig. 5
Fig. 5 Phylogenetic tree of Dof gene family in C.esculentus andother species

Fig. 6
Fig. 6 Cis-elements in promoters of Dofgene family in C.esculentus

Fig. 7
Fig. 7 Transcription factor binding site of Dof gene family in C.esculentus

Fig. 10
Fig. 10 Colinearity relationship analysis between Dofgenes in C.esculentus and other 5 species

Fig. 11
Fig. 11 Interaction protein network of CesDof proteins

Fig. 13
Fig. 13 Gene expression level of CesDof under salt stress by qRT-PCR

Table 1
Physicochemical properties of Dof proteins from C.esculentus

Table 2
Hydrophilicity / hydrophobicity analysis of Dof proteins in C.esculentus

Table 4
The Ka/Ks ratios of CesDof duplication events in C.esculentus Fig. 9 Colinearity analysis between Dof genes in C.esculentus and other species C.esculentus and Rhynchospora pubera.This indicates that Dof gene is more conserved among plants of the same family, and that the genetic relationship between C.esculentus and Rhynchospora pubera is much closer than that of other species.The collinearity relationship of C.esculentus with five other species indicates the following relationships: C.esculentus, Rhynchospora pubera, Rhynchospora breviuscula, Carex littledalei, Rhynchospora tenuis and Arabidopsis thaliana.The closest affinity is between C.esculentus and Rhynchospora pubera, indicating a closer evolutionary relationship.On the other hand, C.esculentus and Arabidopsis thaliana have the furthest affinity, suggesting that Dof plants are more conserved among conspecifics.Conversely, C.esculentus and Arabidopsis thaliana have the farthest genetic relationship.The analysis suggests that Dof plants are more conservative among the same family of plants.From a plant taxonomy perspective, C.esculentus and Carex littledalei, Rhynchospora tenuis and Rhynchospora breviuscula are both Cyperaceae plants with a close genetic relationship.The number of homologous gene pairs between Cyperaceae plants is greater than that between C.esculentus and Arabidopsis, indicating a closer genetic relationship.